Fertility is defined as the capacity of living beings to reproduce. Based on this concept, it is considered that sterility is the loss of this capacity and it is estimated that it affects 15% of couples of reproductive age. In approximately half of cases the male factor is present: in 20% it is exclusively male, in 38% it is predominantly female, and in another 27% it is considered mixed, whereas in the remaining 15% there is no specific cause, and in these cases it is classified as infertility of unknown origin or idiopathic. According to the American Society for Reproductive Medicine (The Practice Committee of The American Society for Reproductive Medicine, 2006), infertility is regarded as a pathology whenever a couple are unable to conceive in a minimum period of 12 months. Nevertheless, between 20% and 30% succeed in having children after this time.
For diagnosis of male infertility, in addition to the main parameters that are determined in semen analysis (sperm concentration, motility and morphology), recently a new parameter has begun to be considered: sperm DNA fragmentation. Analysis of sperm DNA fragmentation determines the existence of breaks in one or both DNA strands. This has attracted some attention because the presence of these breaks compromises an individual's capacity to have healthy children when the paternal genetic message is altered. In fact, in recent years several studies have demonstrated the presence of an increased percentage of sperm with fragmented DNA in infertile individuals relative to fertile individuals (Evenson D P et al. Theriogenology 15: 979-91 (2006)). The effect was immediate in the area of clinical diagnosis of male infertility and it has started to be evaluated as a marker of sperm quality since DNA fragmentation offers a value complementary to the parameters in semen analysis, although it is true that the predictive value with respect to fertility is still under investigation (Zini and Sigman. J Androl. 30(3): 219-29 (2009)).
At present, the values that associate DNA fragmentation with low potential for fertility “in vivo” or “in vitro” are stated to be in the range 30-40% of sperm affected (Evenson D P and Wixon R. Fertil Steril 90(4): 1229-31 (2008)). In these cases, the risk of recurrent abortions, implantation failure or abnormal embryonic development increases significantly (Carrell D T et al. Arch Androl 49 (1): 49-55 (2003)). Conversely, in fertile individuals without other disorders, the percentage of sperm with fragmented DNA is expected to be under 20%, whereas intermediate values between 20% and 30% of fragmentation might indicate an abnormal situation although it could still not be linked to infertility (Erenpreiss J et al. Asian J Androl 8(1): 11-29 (2006)).
The etiology of sperm DNA fragmentation is multifactorial and although the mechanisms that cause these alterations have partly been identified, the origin of this damage is not known with absolute certainty (Tesarik et al. Reprod Biomed Online 12: 715-21 (2006), Angelopoulo R et al. Reprod Biol Endocrinol 5: 36 (2007)). However, at an intrinsic level, it has been suggested that changes during spermiogenesis affect compaction of the sperm nucleus, producing a state of vulnerability to certain forms of oxidative stress that might cause breakage of DNA (Aitken R J and De Iuliis G N. Mol Hum Reprod. 2009 Jul. 31).
Oxidative stress is regarded as one of the main causes of sperm DNA fragmentation. Generally, oxidative stress means that a metabolic imbalance develops in the organ affected, so that the organism is incapable of quickly neutralizing the reactive oxygen species that are produced as a consequence of the constant supply of metabolic energy required for their activity. In this way, as they accumulate they produce damage in all the components of the cell, including the DNA, oxidation of polyunsaturated fatty acids and oxidation of amino acids in proteins.
Various studies have demonstrated that reactive oxygen species, both of endogenous and exogenous origin, can induce sperm DNA breakage in vitro or in vivo, confirming the part played by free radicals in the etiology of male infertility (Iwasaki A et al. Fertil Steril. 1992; 57: 409-16, Zini A Int J. Androl. 1993; 16: 183-8, Tremellen K Reprod Update. 2008 May-June; 14(3): 243-58).
It is estimated that between 25% and 50% of infertile patients have abnormal concentrations of reactive oxygen species (Twigg J et al., Hum Reprod. 1998; 13: 1429-36, Aitken R J et al., Biol Reprod. 1998; 59: 1037-46, Sawyer D E Mutat Res. 2003; 529: 21-34).
In the particular case of patients diagnosed with varicocele, the main disorder that can be corrected surgically, representing between 19% and 41% of cases of infertility, the presence of reactive oxygen species can be even greater compared to other infertile patients (T. Mostafa et al., Andrologia 41 (2009), pp. 125-129, Naughton C K. et al., Hum Reprod Update 7 (2001), pp. 473-481).
In this context, it seems obvious that rational treatment with antioxidant therapies could help to improve the integrity of sperm DNA, since its main effect is directed at maintaining homeostatic equilibrium by neutralizing reactive oxygen species. In fact, several studies have demonstrated a positive result of certain treatments with antioxidants on sperm DNA fragmentation and other important semen parameters such as sperm concentration, motility or morphology (Agarwal A. et al., Reprod Biomed Online. 2004 June; 8(6): 616-27, Greco E. et al., J. Androl. 2005 May-June; 26(3): 349-53, Ménézo Y J. et al., Reprod Biomed Online. 2007 April; 14(4): 418-21). Although there have been few such studies and the sample sizes are insufficient, the data currently available indicate that treatment with oral antioxidants contributes to preserving the integrity of sperm DNA. Ideally, administration of antioxidant treatments should be prescribed after determining the presence of oxidative stress in the patient's sample.
The determination of oxidative stress in semen samples in andrology laboratories is not included in routine practice because the existing methods are expensive, complex and lack standardization.
At present there are about 30 methods for determining oxidative stress (Ochsendorf FR. Hum Reprod Update. 1999 September-October; 5(5): 399-420). These methods are classified as direct methods, indirect methods and sentinel signs.
The direct methods determine the damage produced by the excess of reactive oxygen species against the phospholipids present in the plasma membrane or in DNA. The direct methods determine damage that is the end product of an imbalance between excessive production of free radicals and the cell's antioxidant capacity. This group may include the test for thiobarbituric acid, which requires high-performance liquid chromatography (HPLC) or determination of isoprostane 8-Iso-PGF2a or the c11-BODIPY test. These tests are quite promising but are not used routinely owing to their complexity.
The indirect methods are generally very sensitive and have the advantage that the normal values in fertile and infertile controls are relatively well defined. These methods determine the presence of reactive oxygen species (ROS hereinafter) in semen samples. The ROS include oxygen ions, free radicals and peroxides, both inorganic and organic. They are generally very small, highly reactive molecules that form naturally as a by-product of normal oxygen metabolism and have an important role in cell signaling. They are generally methods based on chemiluminescence using luminol or lucigenin (Athayde K S. et al. J. Androl. 2007, 28: 613-20). However, lucigenin tends to undergo autoxidation, affecting the results, and, furthermore, the analysis requires a luminometer, which is a very expensive instrument. Tunc et al. (Int. J. Androl, 33: 13-21) described an indirect fertility test based on detection of ROS using NBT as indicator. In cells that contain ROS, NBT is converted to formazan, producing a colored precipitate. However, this method has the disadvantage that the sperm in the sample tend to aggregate and sediment in the conditions in which they are incubated to lead to the formation of formazan, which makes it difficult to determine the percentage of sperm that contain ROS.
Finally, there is a set of indicators (sentinel signs) that indicate the presence of oxidative stress, namely: low sperm motility, teratozoospermia, presence of leukocytes in semen, increase in viscosity, HOST test positive or poor membrane integrity.
There is therefore a need to find a method that is economical and easy to carry out, for determining the presence of ROS in a cellular population.